The long-term objectives of this research application is to better understand the cellular and molecular basis of the inflammatory response in order to develop better therapies to augment it in conditions where the host's immune response is compromised, and to inhibit it under conditions where the response is counterproductive, such as in inflammatory diseases like atherosclerosis, rheumatoid arthritis and other autoimmune diseases, septic shock, asthma, and transplant rejection. A critical step in the inflammatory response is the migration of leukocytes out of the bloodstream to the site of inflammation. We have been studying the molecules and mechanisms responsible for diapedesis-the step in this process in which leukocytes pass across the endothelial cells lining postcapillary venules at sites of leukocyte egress. In the first funding period of this grant, we discovered a molecule (CD99) and a mechanism (targeted recycling of membrane from an internal perijunctional compartment) that play significant roles in diapedesis. The aims for the next funding period are to determine how CD99 functions in diapedesis. Specifically, we will examine whether CD99 is in this perijunctional compartment and whether it controls a distinct step in targeted recycling of this compartment around the migrating leukocyte during diapedesis. We have cloned the murine version of CD99 and are developing reagents to block its function. We will use these tools to interfere with CD99 in murine models of acute inflammation to study the role of murine CD99 in the inflammatory response in vivo. While most diapedesis takes place at endothelial cell borders (lateral junctions), under some circumstances leukocytes migrate through the bodies of endothelial cells in a process called emperipolesis. The molecular basis of this is not understood. We have designed an in vitro system in which we can reliably stimulate emperipolesis. We will study the molecular basis of emperipolesis and test the hypothesis that it, like migration at the junctions, involves the targeted recycling of membrane from the perijunctional compartment. We will thus learn more about the regulation of both forms of transendothelial migration.